Estimations of the circadian periods of plasma melatonin, plasma cortisol, and core body temperature rhythms collected under forced desynchrony protocols revealed that the intrinsic circadian period of the human circadian pacemaker was significantly longer following entrainment to the Martian sol as compared to following entrainment to the 23.5-h day. The latter finding of after-effects of entrainment reveals for the first time plasticity of the period of the human circadian timing system. Both findings have important implications for the treatment of circadian rhythm sleep disorders and human space exploration.Human expeditions to Mars will require adaptation to the 24.65-h Martian solar day-night cycle (sol), which is outside the range of entrainment of the human circadian pacemaker under lighting intensities to which astronauts are typically exposed. Failure to entrain the circadian time-keeping system to the desired rest-activity cycle disturbs sleep and impairs cognitive function. Furthermore, differences between the intrinsic circadian period and Earth's 24-h light-dark cycle underlie human circadian rhythm sleep disorders, such as advanced sleep phase disorder and non-24-hour sleep-wake disorders. Therefore, first, we tested whether exposure to a model-based lighting regimen would entrain the human circadian pacemaker at a normal phase angle to the 24.65-h Martian sol and to the 23.5-h day length often required of astronauts during short duration space exploration. Second, we tested here whether such prior entrainment to non-24-h light-dark cycles would lead to subsequent modification of the intrinsic period of the human circadian timing system. Here we show that exposure to moderately bright light (∼450 lux; ∼1.2 W/m The daily pattern of sleep and wakefulness is regulated by homeostatic and circadian processes 2) should be timed close to the circadian core body temperature minimum, which would interfere with normal timing of the sleep episode. In the present study, we therefore evaluated whether exposure to only moderately bright light limited to part of the scheduled wake episode could entrain the human circadian pacemaker at a normal phase angle to the Martian sol and to a 23.5-hour cycle. Given that the resetting effects of light are both phase-dependent 2) for half of each of the scheduled wake episodes  and bedtime and between the 25% dim light melatonin offset (DLMOff25%) and scheduled waketime during the constant posture (CP) protocol following the 23.5-h rest-activity cycles did not significantly change as compared to those phase angles during the baseline CP protocol (25% relative to bedtime and the DLMOff25% relative to scheduled waketime during the CP protocol following the 24.65-h light-dark cycles were significantly later by 1 h and 42 min (P<0.01) and 1 h and 40 min (P<0.05), respectively, as compared to their timing during the baseline CP protocol , plasma cortisol data , core body temperature data , and the composite In this study, we developed a model-based lighting regimen that was effective for entraining individuals at a normal phase angle to the 24.65-h Martian sol and to a 23.5-h rest-activity cycle. Second, we showed for the first time plasticity of the human circadian timing system that regulates period, with prior entrainment to non-24-h rest-activity and light-dark cycles resulting in long-term changes in the intrinsic human circadian period (after-effects of entrainment).We have developed a robust countermeasure composed of appropriately timed moderately bright light, dim light, and darkness, based on the human phase response curve, intensity response curve, and predictions from Kronauer's mathematical model of the effects of light on the human circadian pacemaker Furthermore, here we show that entrainment to a non-24-h sleep-wake cycle can produce an “after-effect” on the intrinsic circadian period in humans and thus demonstrates plasticity of the human circadian timing system. Our finding of after-effects of entrainment in humans demonstrates a fundamental property of the circadian time keeping system in humans that is consistent with that observed in non-humans The magnitude of the after-effects of entrainment in the current study (0.1 h), in which T-cycles that differed from 24 hours by 0.5 and 0.65 hours were imposed on the human subjects for 14 days, was smaller than that observed in studies performed in mice in which a gradually changing T-cycle that differed from 24 hours by ±4 hours was imposed for approximately 50 days Our finding of an after-effect of entrainment on circadian period may help to explain, as least in part, the observed difference in average circadian period between sighted individuals (∼24.2 h) and blind individuals not entrained to the 24-h day while living in society (∼24.5 h), although selection bias and other factors may play an additional role in the longer average period reported in non-entrained blind individuals Also photoperiod, the fraction of the light episode relative to the dark episode within a light-dark cycle, has been reported to affect circadian period in mice and birds, with longer light episodes shortening circadian period. Such differences could account for seasonal variations in circadian timekeeping The findings of circadian entrainment to non-24-h sleep-wake rhythms and after-effects on intrinsic circadian period following entrainment have important implications for the development of treatments for circadian rhythm sleep disorders, such as delayed sleep phase disorder and advanced sleep phase disorder, in which circadian sleep drive occurs much later or earlier than desired, respectively. These findings may also influence development of countermeasures for human adaptation to non-24-h-sleep-wake schedules, as encountered by shift-workers, in jet lag, in submarine naval operations, and during short-term and long-term human space exploration Seven healthy young men (age 22–40 yrs) completed the 73-day in-laboratory studies. Subjects were free from medications and drugs for the week prior and during the study. Subjects reported no shift work for three years nor crossing more than one time zone for three months prior to study. Subjects maintained a regular 8-h sleep and 16-h wake schedule while living at home for three weeks prior to study, as verified by sleep log, call-in times, and wrist actigraphy . Subjects gave written informed consent and the protocol was approved by the Partners Human Research Committee.2] during wakefulness) thereby allowing accurate assessment of intrinsic melatonin phase. After the CP protocol, subjects were scheduled to a 24.65-h or 23.5-h day for 14 cycles, maintaining a 2∶1 wakefulness:bedrest ratio. Given that the resetting effects of light are both phase-dependent 2]) for approximately half of scheduled wakefulness , and dim (∼1.8 lux) for the remainder of scheduled wakefulness. The entrainment schedule was followed by a second 40-h CP protocol and a two-week FD protocol, composed of 28.0-h dim light–dark (∼1.8 lux-0 lux) activity–rest cycles, that was used to estimate the intrinsic circadian period by assessing the subjects' plasma melatonin, plasma cortisol, and core body temperature rhythms Entrainment to non-24-h day lengths and after-effects of entrainment on period were tested in a randomized, within-subject, crossover design. Subjects lived in a personal laboratory room free of time cues for 73 days. Ambient light and room temperature were controlled and sleep opportunities and meals were scheduled. Ceiling-mounted fluorescent lamps  with a 4100-K color temperature produced a spectrum of white light. Napping and exercise were proscribed. After admission to the laboratory, subjects were maintained on a 24-h sleep-wake cycle for three days . This wa125 . The sensitivity of the melatonin assay was 0.7 pg/mL and the interassay coefficient of variation was 13.2% and 8.4% at a mean concentration of 17.3 and 69 pg/mL, respectively. The sensitivity of the cortisol assay was 0.11 µg/dL and the interassay coefficient of variation was 7.9% and 6.8% at a mean concentration of 5.1 and 18.5 µg/dL, respectively. Core body temperature was recorded continuously during the two FD protocols and the five CP protocols every minute via a rectal thermister .Blood was sampled by an intravenous catheter at hourly intervals throughout the FD protocols and CP protocols, and for three 2–3-day sampling windows during the 23.5-h and 24.65-h entrainment cycles. Plasma melatonin and cortisol levels were assayed via radioimmunoassay I25% and DLMOff25% times during the 23.5-h day-night cycle and the 24.65-h day-night cycle, respectively, after exclusion of the first 5 cycles, to minimize transient adjustments to imposed T cycles. The phase angle of entrainment was assessed by comparing the timing of the DLMOn25% and DLMOff25% relative to the scheduled bedtime and scheduled waketime, respectively.To distinguish definitively between entrainment and relative coordination, a much longer inpatient study (>6–12 months) on each non-24-h rest-activity cycle would have been required. We have therefore used a classification method of entrainment that is consistent with that used in our prior research For assessment of the after-effects of entrainment, the intrinsic circadian period following the 24.65-h days and following the 23.5-h days were compared and tested with a 2-tailed t test for dependent samples. Circadian period estimates were based on plasma melatonin, plasma cortisol, and core body temperature data collected during FD and were computed by a nonorthogonal spectral analysis technique with an exact maximum likelihood fitting procedure Text S1Supplementary materials and methods(0.04 MB DOC)Click here for additional data file.Alternative Language Abstract S1Translation of Abstract into Dutch(0.03 MB DOC)Click here for additional data file.


Several parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols.The purpose of this study was to investigate the inter-relationship and the prognostic significance of several biological and clinicopathological parameters in patients with head and neck squamous cell carcinoma (HNSCC) treated by radiotherapy ± chemotherapy.We used two subgroups of a retrospective series for which CT-determined tumoral perfusion correlated with local control. In the first subgroup (n = 67), immunohistochemistry for carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1) was performed on the pretreatment tumor biopsy. In the second subgroup (n = 34), enzyme linked immunosorbent assay (ELISA) was used to determine pretreatment levels of the cytokines vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in serum. Correlation was investigated between tumoral perfusion and each of these biological markers, as well as between the markers mutually. The prognostic value of these microenvironmental parameters was also evaluated.For CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median showed an independent correlation with local control (p = 0.02) and disease-free survival (p = 0.04) with a trend for regional control (p = 0.06).In the second subgroup, IL-6 pretreatment serum level above the median was the only independent predictor of local control (p = 0.009), disease-free survival (p = 0.02) and overall survival (p = 0.005).in vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses.To our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers. Different biological and clinicopathological parameters next to the commonly used tumor and nodal stage have been proposed to help stratifying patients to certain treatment protocols. Specifically, molecular properties of the tumor microenvironment such as hypoxia, inflammation and angiogenesis are currently investigated to serve as a target for therapy.The aim of the present research was to evaluate potential prognostic markers in a retrospective series of HNSCC treated by radiotherapy ± chemotherapy. A recently published report on a retrospective series of patients with a primary HNSCC treated by radiotherapy ± chemotherapy and investigated by dynamic contrast-enhanced perfusion-computer tomography (perfusion-CT), indicated that low tumoral perfusion predicted poor local control (LC), when stratified according to the median perfusion value . This paOur first hypothesis was that CT-determined tumoral perfusion could be a surrogate marker for hypoxia, which is an important negative prognostic factor in radiation treatment of HNSCC ,4.For this purpose, we evaluated the correlation of tumoral perfusion with the expression of carbonic anhydrase IX (CA IX) and glucose transporter-1 protein (GLUT-1) using immunohistochemistry. These antigens are possible intrinsic markers of hypoxia, both mainly controlled by the hypoxia inducible factor-1 (HIF-1) pathway. Their clinical relevance to assess the expression pattern and the relation with hypoxia has been suggested by others for several tumors -8.CA IX is a transmembrane carbonic anhydrase involved in the reversible hydration of carbon dioxide to carbonic acid. It is a widely investigated protein, overexpressed in many tumor types . In HNSCGLUT-1 mediates cellular glucose uptake and thus facilitates anaerobic glycolysis. This protein is largely undetectable in normal epithelium and benign tumors, but is expressed in several types of human carcinomas such as HNSCC and associated with poor prognosis ,7,8,11.Correlation was assessed between tumoral perfusion and each of these intrinsic markers, as well as between CA IX and GLUT-1 mutually. The prognostic value of these immunohistochemical parameters was also evaluated.Our second hypothesis was that the CT-determined tumoral perfusion could be linked with the measurement of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6).VEGF is a pro-angiogenic cytokine known to stimulate the proliferation and migration of endothelial cells and to promote vessel permeability. It is one of the hypoxia-responsive genes, upregulated by increased levels of HIF-1 ,13. VEGFIL-6 is a proinflammatory cytokine produced by inflammatory cells as well as tumor cells ,16. In cAvailable serum samples were analysed to evaluate VEGF and IL-6 at pretreatment time using an enzyme linked immunosorbent assay (ELISA).We intended to evaluate the prognostic value of these serum cytokines, as well as the possible link between tumoral perfusion and the cytokine levels. This link could be established by the hypoxia mediated HIF-1 or NF-κB pathways.The present work results on the possible prognostic significance of different biological and clinicopathological parameters studied in a retrospective study of patients with HNSCC. Furthermore, the relationship with CT-determined tumoral perfusion was investigated.Between March 1995 and January 2000, patients with a primary HNSCC treated by radiotherapy ± chemotherapy were investigated by perfusion-CT to evaluate tumoral perfusion. The largest axial tumor diameter was selected and dynamic CT sequences were taken during injection of a contrast agent bolus. Perfusion was correlated with the carotid artery [The original patient population was divided into two subgroups. For the first subgroup of 67 patients (group A), we had the disposal of formalin-fixed, paraffin-embedded biopsies, taken at the time of diagnostic endoscopy. Serial sections, 5 μm thick, were stained for hematoxylin and eosin (H&E) to allow evaluation of the representativity of the biopsy specimen, and immunohistochemically for CA IX and GLUT-1.For the second subgroup of 34 patients (group B), we had the disposal of serum samples taken at pretreatment time and stored at -20°C until time of ELISA-analyses for VEGF and IL-6. For 24 patients of this subgroup, we had the disposal of other patient-related parameters, such as weight loss, performance status, and sedimentation rate determined at the same pretreatment time.Histological diagnosis was made by an independent pathologist. Clinical data regarding local recurrence, regional recurrence, disease-free and overall survival were available for a minimum follow-up of four years. Patients' characteristics and treatment schedules are shown in Table 2O2 in methanol) was applied for 30 minutes. Slides were rinsed with Phosphate Buffered Saline (PBS) and Phosphate Buffer Saline with 0.4% Tween 20 (PBST) and then incubated with a mouse monoclonal anti-human CA IX antibody  [After dewaxing and rehydrating, an endogenous peroxidase block   for 30 m2O2 in destilled water) was applied for 15 minutes. Slides were rinsed with TBST and incubated with "Protein Block Serum Free" (DAKO) for 20 minutes to block nonspecific binding of immunoglobulins to the tissue. In a next step, sections were incubated with a polyclonal rabbit anti-human GLUT-1 antibody  at 4°C overnight. After 3 × 5 minutes rinses with TBST, incubation with a secondary anti-rabbit labeled polymer/HRP conjugate solution (30 minutes at room temperature) was carried out before rinsing 3 more times. Visualization, counter-staining, dehydrating and mounting was done as for the CA IX staining. Omitting of the primary antibody served as a negative control. To exclude nonspecific binding of the primary antibody, we performed the same protocol using an irrelevant antibody of the same species . This showed no binding (data not shown). Erythrocytes, present in every section, served as internal positive control for GLUT-1. Representative examples of positive and negative staining results are shown in Figure After dewaxing and rehydrating, antigen retrieval was performed by microwaving sections immerged in 10 mM citrate buffer (pH 6). After cooling down, sections were rinsed with Tris Buffered Saline, pH 7,5 with 0.1% Tween 20 (TBST) and an endogenous peroxidase block  per patient.ELISA kits for specific cytokines (VEGF and IL-6) were used according to the manufacturer's protocol . Serum samples were incubated in duplicate on microtiter plates coated with a specific monoclonal antibody for 2 hours at room temperature. The plates were washed to remove unbound antibody. After incubation of a conjugate solution, a substrate solution was added. Color development was stopped after 20–25 minutes, depending on the assay. A microplate reader was used to determine colorimetric densities at 570 nm and 450 nm for each sample. The readings at 570 nm were subtracted from those at 450 nm to get the optical density for each sample. Final results were calculated from a standard curve generated by a form parametric logistic curve fit. Results were expressed in pg/ml.The association between discrete and continuous variables was tested by the Mann Whitney's U-test or Kruskall Wallis test as appropriate. Correlations between continuous variables were obtained using Spearman's rank correlation. Survival curves were constructed using the Kaplan-Meier method, all time intervals being calculated from the date of the first radiotherapy session. Individual factors were evaluated for their predictive value by the log rank test. The Cox proportional hazard model was used for multiple regression analysis. All tests were two-sided, using a significance level of 0.05. All statistics were done using Statistica software 6.On average, the biopsies were composed of viable tumor cells , carcinoma in situ (1.3%), normal epithelium (2.0%), necrotic tissue (2.8%), blood vessels (6.0%), collagenous connective tissue (17.8%), muscle tissue (1.4%), inflammatory connective tissue (14.0%), and a "residu" . Because of their high tumor content the biopsies were considered as representative to score tumoral expression of CA IX and GLUT-1.The CT-determined perfusion rate ranged from 12.4 ml/min/100 g to 274.4 ml/min/100 g with a median value of 87.9 ml/min/100 g (n = 67). The percentage of CA IX positivity ranged from 0 to 87.8% with a median of 17.14%, and the GLUT-1 from 0 to 100% with a median of 67.5%. Both stainings were almost exclusively membranous, except for some aspecific cytoplasmatic CA IX staining.There was no significant correlation between the CT-determined tumoral perfusion and percentage positivity for CA IX or GLUT-1.We also found no significant correlation between tumor characteristics such as tumor stage, nodal stage and differentiation on one hand and perfusion-CT, CA IX and GLUT-1 percentages on the other hand.In univariate analysis, the prognostic value of different clinicopathological and biological parameters towards local control (LC), regional control (RC), disease-free survival (DFS) and overall survival (OS) was evaluated.The tumor stage (T3-4 vs. T1-2) predicted a poorer RC  and OS . N3 stage also seemed to predict poorer outcome . No significant correlation was found between tumor differentiation and outcome.Poorer tumor perfusion stratified according to the median value was linked with poorer LC  and DFS .For the CA IX and GLUT-1 percentages we could not find a significant correlation with outcome. Nevertheless, a trend towards poorer LC (31.4 vs. 44.6% at 2 years p = 0.08) and DFS  was seen in patients with CA IX as well as GLUT-1 values above the median.The multivariate analysis included tumor stage (T3-4 vs. T1-2), nodal stage (N0-1-2 vs. N3), differentiation grade, perfusion (≥ median) and combined CA IX and GLUT-1 percentages (CA IX and GLUT-1 ≥ median).As shown in Table The CT-determined tumoral perfusion rate ranged from 29.6 ml/min/100 g to 263.6 ml/min/100 g with a median value of 88.7 ml/min/100 g (n = 34). IL-6 values ranged from 0.4 pg/ml to 57.3 pg/ml with a median of 5.4 pg/ml. VEGF values ranged from 73.5 pg/ml to 1613.1 pg/ml with a median of 326.9 pg/ml.There was no significant correlation between the perfusion-CT measurements, IL-6 and VEGF values. However, Figure Patients with higher nodal stage showed significantly more elevated IL-6 serum levels (p = 0.04). Other tumor characteristics such as tumor stage and differentiation grade were not correlated with either perfusion-CT, serum VEGF or serum IL-6 levels.In univariate analysis, the prognostic value of IL-6, VEGF and clinicopathological parameters towards LC, RC, DFS and OS was evaluated.As clear from Figures The multivariate analysis included tumor stage (T3-4 vs. T1-2), nodal stage, perfusion (≥ median), VEGF serum level (≥ median) and IL-6 serum level (≥ median). Table The tumor microenvironment plays a critical role in tumor aggressiveness and treatment response. Several microregional features have been shown to be relevant for treatment outcome, such as hypoxia, inflammation and angiogenesis. The major question stays whether these markers can be used in an integrated way to stratify patients to certain treatment options .Starting from a retrospective series of patients with HNSCC treated by radiotherapy ± chemotherapy, we investigated the prognostic significance of several biological and clinicopathological parameters. Earlier established results showed that for this whole patient group, CT-determined tumoral perfusion was correlated with poorer LC .Two subgroups were now used for further analysis. In the first subgroup of 67 patients, immunohistochemistry for CA IX and GLUT-1 was performed on a representative paraffin-embedded pretreatment biopsy. In the second subgroup of 34 patients, ELISA was used to determine pretreatment VEGF and IL-6 serum levels. The correlation between these parameters mutually, as well as their prognostic value, was investigated.Hypothesizing that CT-determined tumoral perfusion could be an inverse marker for hypoxia (less perfusion relates to more hypoxia), we assessed the correlation between tumoral perfusion, the intrinsic hypoxia markers CA IX and GLUT-1, and the serum levels of VEGF and IL-6, known to be upregulated under certain microenvironmental conditions like hypoxia.Besides a borderline inverse association between perfusion and VEGF, no significant correlation was found in our study. There are three possible explanations in support of this finding.First, it is likely that the dynamic CT measures rather acute hypoxia, while CA IX and GLUT-1, as well as VEGF and IL-6, are more indicative for chronic hypoxia.Second, the CT-determined tumoral perfusion in this patient group was a measurement that took place through one region of interest, i.e. the maximum diameter of the tumoral region. On the other hand, CA IX and GLUT-1 immunohistochemistry was performed on one tumor biopsy taken at the periphery of the tumor field, and VEGF and IL-6 values were measured as systemic serum levels.Third, the relationship between perfusion and oxygenation has shown to be in a precious balance ,25.The borderline association found between perfusion and VEGF can be explained through the hypoxia-hypothesis. Nevertheless, an opposite finding would also have been possible, associating the angiogenetic property directly with perfusion.The further analysis of the biological and clinicopathological parameters showed that patients with higher nodal stage expressed significantly higher IL-6 serum levels. This experience is in accordance with previous reports linking elevated IL-6 levels to extensive disease , and morConcerning the prognostic value of the clinicopathological parameters, we found a correlation with outcome for known factors like tumor stage (RC and OS) and N3 stage . Of these, only the nodal stage appeared to behave as an independent prognostic factor for RC and DFS.Concerning the prognostic value of the biological parameters, poorer tumor perfusion predicted poorer LC and DFS, as expected . This coFor CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median did indicate an independent correlation with worse LC, RC and DFS. This result demonstrates that a combined assessment gives additive information, though we have to note that the prognostic value of each of both markers is not clearly established, given prognostic -8,27 as In the second subgroup, IL-6 pretreatment serum level stratified according to the median seemed to be the only independent predictor of LC, DFS and OS. VEGF pretreatment levels were not correlated with prognosis.in vitro by others [The prognostic value of IL-6 has been described in many tumor types , but noty others -23.VEGF- reports in literature are a bit conflicting, with papers demonstrating  and denyIn this study, due to methodological reasons, we performed serum and immunohistochemical analyses for two subgroups instead of the whole patient group.The advantage of using serum samples over immunohistochemistry on biopsies is that the ELISA test is quickly and simple to perform. Furthermore, the technique is not too invasive and quite established.On the other hand we must be aware that HNSCC cells may not be the only source of the elevated serum cytokine levels in patients. The serum levels may also depend in part upon individual host inflammatory responses and conditions like cachexia, and certainly not upon hypoxia only. In our patient group however, we did not see a correlation between VEGF or IL-6 serum levels and sedimentation rates, determined at the same pretreatment time. Furthermore, there are some pitfalls in the measurement of these cytokines . Concernin vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses. This finding has to be validated in other, preferably prospective, studies.In conclusion, we found a prognostic value in this series of patients with HNSCC treated by radiotherapy ± chemotherapy for some of the investigated parameters, with a major role for IL-6. To our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers.We believe that additional to the standard TNM classification this combined assessment of several biological and clinicopathological parameters will show most appropriate to stratify patients to certain treatment protocols. The efforts made to search for prognostic factors in radiotherapy settings will stretch towards a more efficient combination of radiotherapy with other treatment strategies like chemotherapy, radiosensitizers, bioreductive drugs and biological targeting. Therefore, further research will be necessary.The author(s) declare that they have no competing interests.HDS carried out the immunohistochemistry, ELISA's and the acquisition and analysis of data. She also drafted the manuscript. WL participated in the conception of the study and helped to draft the manuscript. EV assisted in the immunohistochemistry performance and scoring technique. LG participated in the scoring process, and the analysis of data. RH performed perfusion-CT and collected data. SN conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Epidemiological evidence strongly links fish oil, which is rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with low incidences of several types of cancer. The inhibitory effects of omega-3 polyunsaturated fatty acids on cancer development and progression are supported by studies with cultured cells and animal models. Propofol  is the most extensively used general anesthetic–sedative agent employed today and is nontoxic to humans at high levels (50 μg/ml). Clinically relevant concentrations of propofol  have also been reported to have anticancer activities. The present study describes the synthesis, purification, characterization and evaluation of two novel anticancer conjugates, propofol-docosahexaenoate (propofol-DHA) and propofol-eicosapentaenoate (propofol-EPA).The conjugates linking an omega-3 fatty acid, either DHA or EPA, with propofol were synthesized and tested for their effects on migration, adhesion and apoptosis on MDA-MB-231 breast cancer cells.At low concentrations (25 μM), DHA, EPA or propofol alone or in combination had minimal effect on cell adhesion to vitronectin, cell migration against serum and the induction of apoptosis (only 5 to 15% of the cells became apoptotic). In contrast, the propofol-DHA or propofol-EPA conjugates significantly inhibited cell adhesion (15 to 30%) and migration (about 50%) and induced apoptosis (about 40%) in breast cancer cells.These results suggest that the novel propofol-DHA and propofol-EPA conjugates reported here may be useful for the treatment of breast cancer. Included in the growing list of affected cell lines are breast cancer cells [Omega-3 polyunsaturated long-chain fatty acids (ω-3 PUFAs) have been documented to inhibit or even prevent cancer. Epidemiological evidence strongly links fish oil (rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) with low incidences of several types of cancer -6. The ier cells ,8,15-17.er cells ,19 and ier cells . Propofoer cells -24 and her cells ,26, inhier cells  and increr cells . Propofoc [Although most studies concerning the mode of action of propofol have concentrated on its action as an anesthetic, there are a few reports indicating that this compound may also affect cellular processes related to cancer. Clinically relevant concentrations of propofol (3 to 8 μg/ml) were reported to decrease the metastatic potential of human cancer cells, including HeLa, HT1080, HOS and RPMI-7951 cells . In addic . The conThe omega-3 PUFAs DHA and EPA are natural nontoxic food substances that have interesting anticancer properties. Propofol is a widely employed nontoxic anesthetic that also has anticancer properties. Although several preparations of propofol with lipid mixtures are available for anesthetic usage , to our knowledge propofol has not previously been covalently conjugated with fatty acids. The purpose of this study was to synthesize and investigate novel compounds composed of DHA or EPA conjugated to propofol. The conjugates were tested for their ability to inhibit breast cancer cell migration, alter adhesion to the matrix protein vitronectin and induce apoptosis. The results indicate that these novel conjugates might represent a new class of anticancer agents.N,N-dicyclohexylcarbodiimide, 2,6-di-tert-butyl-4-methylphenol (BHT), 4-(dimethyl amino)pyridine, hematoxylin, crystal violet and all other reagents were from Sigma Chemical Co. .MDA-MB-231 breast cancer cells were purchased from the American Type Culture Collection . The Vybrant Apoptosis assay kit was from Molecular Probes , and DMEM, penicillin, streptomycin and glutamine were from Invitrogen Corporation . Fetal bovine serum was from BioWhittaker . Transwell chemotaxis chamber plates were from Corning Incorporated . Cytomatrix Human Vitronectin-Coated Strips were from Chemicon International, Inc. . DHA and EPA fatty acid standards for thin-layer chromatography (TLC) and gas chromatography (GC) were from Nu-Chek Prep, Inc. . Methanol, chloroform, petroleum ether, diethyl ether, acetic acid, hexane and ethanol were from Fisher Scientific . Propofol, N,N-dicyclohexylcarbodiimide (0.45 mmol), and an antioxidant, BHT (1.5 μM), were dissolved in 5 ml of chloroform and the reaction was stirred for 60 min at room temperature (23–25°C). To this mixture, propofol (0.29 mmol) and 4-(dimethyl amino)pyridine (0.152 mmol) were added. The mixture was stirred for a period of 12 hours; the suspension was then filtered, washed with light petroleum (38.3–53.2°C) and subjected to purification on analytical thin-layer plates . The plates were developed in a solvent mixture of petroleum ether and ethyl acetate  and the products were revealed with iodine vapor. The spots were compared with authentic DHA, propofol, BHT, pyridine and N,N-dicyclohexylcarbodiimide and the spot corresponding to the new compound was scraped, suspended in chloroform/methanol , passed through a glass filter and stored at -70°C. The propofol-DHA and propofol-EPA conjugates were characterized as described below.Although only propofol-DHA synthesis is described here, the procedure is analogous for the propofol-EPA conjugate. Synthesis was performed in two steps. First, docosahexaenoic acid anhydride (DHA-anhydride) was synthesized, followed by its esterification to propofol. Synthesis of the conjugate was performed under reduced light and under nitrogen to minimize auto-oxidation. In brief, DHA or EPA (0.300 mmol), a coupling reagent, Characterization of the propofol-DHA conjugate was performed by a combination of techniques. First, the presence of propofol in the conjugate was assessed by UV spectroscopy with an SLM Aminco 3000 spectrophotometer. The conjugate was dissolved in ethanol and the absorption spectra was measured from 200 to 600 nm. Next, the new compound was hydrolyzed and methylated in 3 M methanolic HCl . The pro6 cells/ml for routine culture. For experimental purposes, cells were cultured at the cell density indicated and treated with DHA, EPA, propofol or the conjugates. The test compounds were stored in hexane at -80°C. An aliquot of the conjugate was dried under nitrogen and the compounds were diluted in ethanol just before use. The final concentration of ethanol (less than 0.1%) in the treated cultures did not exhibit any cytotoxic effects as measured by lactate dehydrogenase release and a WST-cell proliferation assay (results not shown).MDA-MB-231 breast cancer cells were grown in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin at a density of 10The effect of the fatty acids on cell growth was determined with a WST-1 assay in accordance with the manufacturer's instructions .5/ml) in serum-free DMEM was added to the top chamber with or without (control) test compounds. The plates were incubated for 4 hours at 37°C in a humidified CO2 incubator. After incubation, the insides of the inserts were washed and cleaned with cotton swabs, and the filters were fixed with 5% formaldehyde. The cells were stained with hematoxylin as described [Cell migration was performed with Transwell Chemotaxis Chamber Plates (Corning Inc.). The bottom chamber was supplemented with 300 μl of DMEM containing 10% fetal calf serum, and 100 μl of MDA-MB-231 cells (10escribed  and cell5/ml) at 37°C for 45 min in a CO2 incubator under serum-free conditions with control or test compounds. The wells were washed three times with PBS and the adhered cells were stained with 0.2% crystal violet in 10% ethanol for 5 min at room temperature. The excess stain was removed by washing six times with PBS. The stained cells were dissolved in 100 μl of solubilization buffer  and the absorbance was read at 540 nm. Absorbance of dye in the control (vehicle-treated) cells was regarded as 100% adherence and the percentage adherence of treated cells was calculated in comparison with that of the control cells.The cell adhesion assay was performed with Cytomatrix human vitronectin-coated strips . The strips were incubated with 100 μl of MDA-MB-231 cells (105/ml) suspended in serum-free DMEM were incubated in 96-well plates at 37°C in a humidified CO2 incubator with control or test compounds. At the end of the incubation period (24 hours), reagents A  and B  from the kit were added to each well (1 μl/ml) and the plates were left to incubate for 30 min on ice. The cells were revealed by using a fluorescence microscope with appropriate filters. Live cells do not exhibit any fluorescence because the dyes are impermeable to living cells, dead and necrotic cells exhibit red fluorescence, and apoptotic cells fluoresce green. Total and apoptotic cells were counted and the percentage of cells exhibiting apoptosis was calculated.A Vybrant apoptosis assay kit (Molecular Probes) was used in accordance with the manufacturer's protocol. In brief, 100 μl of MDA-MB-231 cells (105 per well) were grown in 48-well plates in serum-free DMEM for 24 hours at 37°C in a humidified CO2 incubator with control or test compounds. After incubation, a caspase-3 fluorescent substrate ) was added to each well (10 μl per well) and the plates were incubated for a further 1 hour. Cells were revealed under a fluorescence microscope and pictures were taken with a MagnaFire charge-coupled device camera .Activation of caspase-3 in MDA-MB-231 cells was determined with a caspase-3 activity assay kit . Cells  were grown in 48-well plates in serum-free DMEM for 24 hours at 37°C in a humidified CO2 incubator with control or test compounds. Cells were then homogenized in a buffer containing 10 mM HEPES, pH 7.4, 0.25 mM sucrose and 1 mM EDTA; a post-nuclear fraction was prepared by centrifugation at 2,000 g for 5 min at 4°C. The supernatant was further centrifuged at 100,000 g for 20 min at 4°C and the resultant cytosolic fraction was used for cytochrome c detection by immunoblotting. Proteins were separated by 8 to 15% SDS-PAGE and the blot was incubated with monoclonal anti-cytochrome c  or monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase  overnight at 4°C and detected with secondary anti-mouse peroxidase-conjugated antibodies . The bands were detected with a chemiluminescence detection kit . The relative distributions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase (loading control) were determined by densitometric analysis with the Kodak imaging system .Induction of apoptosis was also assayed by detecting cytochrome escribed . Cells .RF of 0.90. The RF of this product is very different from that of either DHA or propofol. The band corresponding to the new compound was scraped and subjected to spectrometric characterization. The propofol spectra showed two absorption peaks, at 216 and 274 nm; these peaks were shifted to 205 and 265 nm, respectively, for the propofol-DHA conjugate (data not shown). Next, the conjugate was hydrolyzed and then methylated for analysis by gas chromatography. The results demonstrate that the commercially available propofol  was 97% pure, and analysis of the hydrolyzed product of the propofol-DHA conjugate showed a DHA : propofol ratio of 1: 0.8. The ratio varied from the expected 1:1 because other isoforms of propofol were also conjugated to DHA (data not shown).The chemical synthesis of the propofol-DHA conjugate is shown in Fig. -1, which are attributable to C = O and C–O bonds, respectively, indicating the presence of an ester. The band at 3,030 cm-1 is characteristic of an aromatic C–H bond (propofol), and the band at 2,800 to 2,960 cm-1 is characteristic of aliphatic C–H bonds. No O–H absorption band was seen, indicating the absence of non-esterified propofol. The presence of an ester bond, aromatic C–H absorbance, and absence of free O–H group absorbance further confirms the formation of a propofol-DHA conjugate.Further characterization of the conjugate was performed by infrared spectroscopy. The infrared absorption spectra of the propofol-DHA conjugate Fig.  showed tThe propofol-EPA conjugate was also synthesized; its characterization by TLC, UV spectroscopy, GC analysis of hydrolyzed product and infrared spectroscopy was performed in a similar fashion (data not shown).Finally, a mass spectroscopic analysis was performed to determine the molecular mass of the product on a MAT 95XP high-resolution, high-mass-accuracy mass spectrometer. Its high mass accuracy (less than 5 p.p.m. in magnetic scan and less than 2 p.p.m. in electric scan) enables its data to be used to provide a formula match. That is, the mass spectra are precise enough to verify a chemical formula based on the sum of the mass defects from the constituent atoms. Results demonstrate that the propofol-DHA mass spectra identified a product with a parent molecular mass of 488.36 Da, which is very close to the calculated molecular mass, 488.74 Da. The observed monoisotopic molecular mass was within 0.8 part per million (0.0004 atomic mass units) of that predicted for propofol-DHA. The propofol-EPA spectra identified a product of molecular mass 462.34 Da, which is very close to the calculated molecular mass of 462.70 Da. The observed monoisotopic molecular mass was within 1.1 parts per million (0.0006 atomic mass units) of that predicted for propofol-EPA .Characterization of the propofol-DHA conjugate and propofol-EPA conjugates suggests that the coupling reaction between DHA or EPA and propofol resulted in the formation of new products. The conjugates are a one-to-one ester of DHA or EPA and propofol.Results shown in Fig. P < 0.05).Because MDA-MB-231 breast cancer cells are highly invasive, we monitored the effect of the conjugates on cell migration. The results shown in Fig. P < 0.05) and 30% (P < 0.05), respectively.We also tested the effect of the propofol-DHA and propofol-EPA conjugates on adhesion of the breast cancer cells to a vitronectin substrate. DHA and EPA alone did not significantly affect breast cancer cell adhesion, whereas propofol itself or in combination with DHA or EPA slightly increased cell adhesion Fig. . In contP < 0.05) of the breast cancer cells. To confirm the apoptotic response in these cells, we further assayed for caspase-3 activity, a protease involved in the execution phase of apoptosis. The assay was performed by using a specific fluorescent caspase-3 substrate (DEVD-AFC) that on hydrolysis produces a green fluorescent product. The results shown in Fig. Propofol-DHA and propofol-EPA were tested for their ability to initiate apoptosis within MDA-MB-231 breast cancer cells. Induction of apoptosis was assayed by incubating the cells with DHA, EPA or propofol alone or mixtures of DHA and EPA with propofol as well as with the propofol-DHA and propofol-EPA conjugates. Results shown in Fig. c release  increase in cytochrome c release. However, propofol-DHA or propofol-EPA conjugates caused significant increases in cytochrome c release by about 300 to 400% (P < 0.05).The effect of propofol-DHA and propofol-EPA conjugates on apoptosis was further analyzed by assaying for cytochrome ase Fig. . DHA, EPThese results confirm that the propofol-DHA and propofol-EPA conjugates are far more effective at inducing apoptosis in breast cancer cells than are the unconjugated parent compounds DHA, EPA or propofol.Often a major obstacle to the successful use of a drug is its ability to be taken up and retained by cells. Either the drug must have its target on the outer membrane surface or it must cross the plasma membrane through either an existing transport system or by simple diffusion to affect intracellular targets. One approach to overcoming the problem of cell entry and retention has been to link water-soluble drugs to lipophilic carriers. Several attempts have been made in the past to synthesize novel compounds by conjugating fatty acids with drugs. For example, chlorambucil-fatty acid conjugates (Chl-fatty acid) were synthesized and tested on human lymphoma cell lines . These sSimilarly, DHA-paclitaxel was synthesized by Bradley and colleagues, who demonstrated that the conjugate possessed antitumor activity in mice with lung tumors . In the sn-2 position and either stearic acid or DHA in the sn-1 position. The DHA-containing and methotrexate-containing phosphatidylcholines were more effective than conjugates containing stearic acid. Synthesis of phosphatidylcholines containing DHA and propofol is also a possibility, but such a synthesis is more cumbersome. In the study described here, we therefore used another approach to synthesize a class of novel compounds by directly conjugating DHA or EPA with propofol. Results presented in Figs We have previously employed the DHA-conjugate approach to enhance the availability and hence the activity of the anticancer drug methotrexate . Two phoAfter obtaining the conjugates, we investigated their anticancer effects on breast cancer cells. MDA-MB-231 is a highly invasive breast cancer cell line that has the potential for uncontrolled proliferation and metastasis in animal models . We invec release . These conjugates were not tested in other breast cancer cell lines or other cancers of different anatomical locations. Experiments are under way to test these conjugates on different cancer cells lines and also in model systems BHT = 2,6-di-tert-butyl-4-methylphenol; Chl = chlorambucil; DEVD-AFC = Asp-Glu-Val-Asp-fluoromethylketone-aminotrifluoromethylcoumarin conjugate; DHA = docosahexaenoic acid; DMEM = Dulbecco's modified Eagle's medium; EPA = eicosapentaenoic acid; GC = gas chromatography; ω-3 PUFAs = omega-3 polyunsaturated fatty acids; TLC = thin-layer chromatography.The author(s) declare that they have no competing interests.RAS, GZ, and SW designed conjugates, supervised experiments and contributed in manuscript writing. MZ led the propofol-DHA and propofol-EPA synthesis and characterization. MW, AC, and KH were responsible for laboratory work for adhesion, migration and apoptosis assays. All authors read and approved the final manuscript.A Word document containing mass spectrometry data for propofol-DHA and propofol-EPA conjugates are presented as Click here for file


The objective of this study was to evaluate serum IGF-I levels in postmenopausal women with breast cancer treated primarily with raloxifene.Twenty-two postmenopausal patients with operable, stage I or II, estrogen receptor-positive carcinomas participated in this study. Following confirmation of diagnosis, the patients received 60 mg of raloxifene for 28 days prior to definitive surgery. Blood samples were collected for evaluation of serum IGF-I levels prior to initiating medication and following a 28-day treatment course. Student's t-test for paired samples was used in the statistical analysis. Significance was established at p < 0.05.Mean serum IGF-I levels pre- and post-raloxifene treatment were 143.7 ± 9.7 ng/ml and 94.8 ± 7.6 ng/ml, respectively. This reduction in serum IGF-I levels following treatment with raloxifene was statistically significant (p < 0.001).Raloxifene significantly reduced serum IGF-I levels in postmenopausal women with breast cancer. Insulin-like growth factor-I (IGF-I) is produced principally in the liver upon stimulation by GH and plays an important role in promoting normal and neoplastic cell proliferation -8. InsulThe IGF system is currently recognized as a risk factor for the principal types of epithelial cancer ,12. StudSignificantly reduced serum IGF-I levels have also been reported in postmenopausal women with breast cancer treated primarily with tamoxifen ,3. HowevTo the best of our knowledge, only one study published in the literature has evaluated the effects of raloxifene, administered for a period of 14 days prior to surgery, on serum IGF-I levels as a primary treatment for breast cancer in postmenopausal women . It is pThe protocol of this study received the approval of the Institutional Review Board of the Federal University of Piauí. All volunteers gave their signed, informed consent prior to initiation of the study. Twenty-two postmenopausal women in amenorrhea for at least two years, who had sought medical care at the Mastology Department of the Federal University of Piauí and who had been diagnosed with operable, estrogen-receptor-positive, invasive ductal carcinoma for which they had received no prior treatment, were enrolled to this study. Following hematoxylin-eosin staining and confirmation of the diagnosis of invasive ductal carcinoma, the paraffin blocks containing the samples underwent histochemical analysis to evaluate estrogen receptor status. Tumors with nuclear staining measured semiquantitatively as high (>10% immunoreactive cells) were considered positive. Patients with endocrinopathies, those in use of hormonal medication or any other medication that could interfere with serum IGF-I levels were excluded from the study. Tumors ranged from 2 to 5 cm in size, stages I or II. Patients were aged 47 to 87 years (mean 63 years).The patients received 60 mg of raloxifene/day for a period of 28 days prior to definitive surgery, starting at the time of confirmation of the diagnosis following tumor biopsy.For the analysis of serum IGF-I levels, two fasting blood samples were taken, one at baseline, i.e. prior to the initiation of raloxifene therapy, and the second after a 28-day course of the treatment. Plasma was separated by centrifugation and aliquots were stored at -20°C until assayed. All tests were carried out by a professional who was blinded with respect to patient identification.For the analysis of the serum concentrations of insulin-like growth factor-I, the automated immunoassay analyzer, DPC Immulite 2000  was used, and methodology consisted of a solid-phase enzyme-labeled chemiluminescent immunometric assay. Analytic sensitivity was 20 ng/ml and the reference values varied according to age.Comparison of the means of serum IGF-I levels measured prior to and after 28 days of raloxifene use was carried out using Student's t-test for paired samples. Statistical significance was established at p < 0.05.Mean pretreatment serum IGF-I level was 143.7 ± 9.7 ng/ml, whereas mean post-treatment serum IGF-I level was 94.8 ± 7.6 ng/ml  axis in carcinogenesis has grown following the finding of elevated serum levels of insulin-like growth factor-I in association with the principal forms of epithelial cancer ,12. In a


Not much is known about the role of COMP in human cartilage tissue in vivo. With the help of immunohistochemistry, Western blot, in situ hybridization, and real-time reverse transcription-polymerase chain reaction, we aimed to elucidate the role of COMP in human embryonic, adult healthy, and osteoarthritis (OA) cartilage tissue. COMP is present during the earliest stages of human limb maturation and is later found in regions where the joints develop. In healthy and diseased cartilage tissue, COMP is secreted by the chondrocytes and is often associated with the collagen fibers. In late stages of OA, five times the COMP mRNA is produced by chondrocytes found in an area adjacent to the main defect than in an area with macroscopically normal appearance. The results indicate that COMP might be involved in human limb development, is upregulated in OA, and due to its wide binding repertoire, could play a role in the pathogenesis of OA as a factor secreted by chondrocytes to ameliorate the matrix breakdown.As a member of the thrombospondin gene family, cartilage oligomeric protein (COMP) is found mainly in the extracellular matrix often associated with cartilage tissue. COMP exhibits a wide binding repertoire and has been shown to be involved in the regulation of chondrogenesis  COMP hain vitro  and collin vitro ,11. In cin vitro . The DNAin vitro  and alsoin vitro . COMP exin vitro . The humosome 19 . Mutatioosome 19 -18. Furtosome 19  and osteosome 19 ,21. Duriosome 19 , and durosome 19 . Fang anosome 19  detectedevidence , has sugevidence . There aevidence . We therAborted human embryos were obtained according to the regulations of the Ethics Committee of the Medical Faculty of the University of Göttingen, Germany. The embryos were classified as follows: three embryos of gestational week (gw) 8, three embryos of gw 10, and three embryos of gw 12. The ages were determined from histological data according to Carnegie stages . No malfAdult human articular cartilage from the knee joint was obtained from 12 patients (ages 55–75) with OA who were undergoing total knee replacement operations. The patients met the American College of Rheumatology classification criteria for OA of the knee . All pat3) were embedded in LR-Gold®  according to standard procedures, and ultra-thin sections were cut with a Reichert's ultramicrotome and collected on nickel grids coated with Formvar® .The abortion material and the cartilage specimens were transported to the laboratory in histidine-tryptophane-ketoglutarate solution at 4°C to ensure good preservation of the tissues . The samThe anti-COMP antibody is a polyclonal rabbit-anti-bovine antibody that has been affinity-purified . AffinitHealthy cartilage and OA cartilage samples from the area adjacent to the main defect were pulverized. Proteins were extracted using 5 M guanidine hydrochloride and protease inhibitors NEM , EDTA, benzamidine hydrochloride, and amino caproic acid, precipitated in ethanol, washed in PBS, precipitated again, and finally dissolved in PBS containing 0.4% SDS. All experiments were carried out under reducing and denaturing conditions. Protein separation was performed applying SDS-PAGE and using systems containing 6% acrylamide in stacking gels and 12% in the separation gel. Tris-glycine was applied as electrophoresis buffer, and separation was carried out at 100–120 V.After the electrophoresis, the proteins were blotted onto nitrocellulose membranes. Transfer was controlled by Ponceau S staining. Thereafter, membranes were washed until no color was left and then blocked overnight in PBS + 10% (w/v) milk powder at room temperature. Immunoreactions were performed applying the anti-COMP antibody for 2 hours, diluted 1:100 in PBS. The secondary goat-anti-rabbit antibody coupled to alkaline phosphatase was diluted 1:500 and incubated for 1 hour at room temperature. Three 5-minute washes with PBS were carried out between all incubation steps. Visualization was achieved using NBT/BCIP (nitrobluetetrazoline chloride/5-bromo-4-chloro-3-indolyl toluidine) coloring agent .202 in the dark. Each of the reactions was followed by rinsing for 10 minutes in PBS. The sections were pre-treated for 5 minutes with 10 μg/ml protease XXIV . The anti-COMP antibody was applied at a dilution of 1:100 in PBS for 1 hour at room temperature. A standard peroxidase-anti-peroxidase procedure followed, applying a peroxidase-coupled goat-anti-rabbit antibody  at a dilution of 1:150 in PBS for 1 hour at room temperature. The color reaction was carried out with DAB (diaminobenzidine) substrate (Sigma).Immunoperoxidase staining was performed on paraffin-embedded tissue sections as follows: The tissues were deparaffinized, rehydrated, and rinsed for 10 minutes in PBS. Endogenous peroxidase was inhibited by a 45-minute treatment with a solution of methanol and 3% HAs negative controls, each immunoreaction was accompanied by a reaction omitting the primary antibodies and applying rabbit serum diluted 1:100 in PBS instead. All controls proved to be negative.As secondary antibody, an anti-rabbit immunoglobulin G (IgG) (Medac) was labeled with gold particles according to standard procedures. Ultrathin tissue sections were incubated with the anti-COMP antibodies diluted 1:100 in PBS for 16 hours at room temperature. The secondary gold-coupled antibodies, diluted 1:300 in PBS, were applied for 20 minutes at room temperature. Staining with uranyl acetate followed, and reactions were examined with the help of a Zeiss EM Leo 906E electron microscope .The grids were incubated with pure gold solution in order to exclude unspecific binding of free colloidal gold. Furthermore, the reactions were performed with gold-coupled goat-anti- rabbit IgG, omitting the primary antibody to exclude non-specific IgG binding.3shareware [In vitro transcription of non-radioactive sense and antisense RNAs with a DIG labeling kit  was performed applying SP6- and T7-polymerases . After extraction of the probes with phenol-chloroform, these were precipitated with absolute ethanol and the pellet was dissolved in DEPC-H2O (diethyl-pyrocarbonate).RNA was isolated as described below and reverse-transcribed into COMP-specific cDNA. Polymerase chain reaction (PCR) was performed with primers specific for COMP (forward AGGGAGATCGTG CAGACAA and reverse AGCTGGAGCTGTCCTGGTAG) to generate a 154 bp product. They were designed with the help of the primerhareware . CorrespFor light microscopic investigations, paraffin sections were deparaffinized, rehydrated, and pre-treated with proteinase K. The probe concentration was 100 ng of DIG-labeled antisense probes in 100 μl hybridization solution  for each section. Hybridization was carried out for 18 hours at 45°C. Posthybridization treatment included a washing procedure with 2 × SSC , 1 × SSC , 0.1 × SSC  and 0.05 × SSC . Afterward, the incubation with the anti-DIG peroxidase-labeled antibody diluted 1:300 in PBS was started. Finally, color reactions were started with AEC (3-amino-9-ethylcarbazol) substrate. For electron microscopy, nickel grids were incubated for 19 hours at 50°C with the same hybridization solution as described above. The probe concentration was 100 ng of DIG-labeled antisense probes in 20 μl hybridization solution per grid. Rinsing steps were the same as described above. Afterward, sections were incubated with a gold-coupled anti-DIG antibody in PBS (diluted 1:60) for 1 hour at room temperature. The specimens were rinsed with PBS, contrasted, and analyzed with the Zeiss EM Leo 906E.Each of the hybridizations was accompanied by one with an equivalently labeled amount of sense probe. Furthermore, hybridizations were performed without any RNA probes. Additionally, for the ultrastructural controls, tissue sections were treated with pure gold solution or the coupled anti-DIG antibody alone.in situ hybridization at the ultrastructural level, randomly chosen micrographs of cartilage tissue with a normal appearance which were taken from the lateral aspects of a condyle and tissue samples taken from the area adjacent to the main defect from OA cartilage (n = 10) were pooled and counted for gold particle contents. Mean values of the numbers of gold particles per cell were analyzed in the area of 5,000 nm2 in 10 cells from each patient. Significant differences in the number of gold particles were noted for p values (p ≤ 0.01), using the Wilcoxon-Mann-Whitney test for unpaired samples.For ® and RNeasy kit , following the manufacturer's instructions, and then treated with DNAfree® . The quality of the RNA was tested with an Agilent 2100 Bioanalyser RNA chip . The RNA was reverse-transcribed with the help of the Advantage® RT-for-PCR kit  by applying Moloney Murine Leukemia Virus reverse transcriptase and oligo-(dT)18-primer.Pieces (2 mm thick) of OA cartilage tissue taken from the area adjacent to the main defect and pieces of tissue with a macroscopically normal appearance of the lateral aspect of a condyle from each of the 12 patients were minced, and RNA was isolated according to a protocol combining TrizolHPRT-1 (NM_000194) as housekeeping gene and for COMP. The PCR was performed in a total volume of 50 μl with 150 ng cDNA, 5 μl 10× reaction buffer, dNTP 10 μmol each, 20 pmol of each primer, and 2.5 U HotStarTaq® DNA polymerase (Qiagen) with the DNA engine Opticon™ 2. After an initial activation step of 15 minutes at 95°C, further steps were as follows: 35 cycles of denaturing 30 seconds at 94°C, annealing 30 seconds at 61°C, elongation for 30 seconds at 72°C, and (lastly) extension of 10 minutes at 72°C. Ten microlitres of each sample were loaded onto a 1.5% agarose gel and were visualized by ethidium bromide after electrophoresis.PCR conditions were optimized by applying the gradient function of the DNA engine Opticon™ 2  for 2 concentration was determined. Twelve point five microlitres of 2xQuantiTect™ SYBR® Green PCR Master Mix (Qiagen), 20 pmol of each primer, and 250 ng of cDNA were added to a final volume of 25 μl. Cycling was performed with the protocol described above. Data acquisition was carried out after each extension step, and a melting curve was performed in 0.1°C steps from 50°–95°C. Real-time RT-PCR efficiencies were calculated from the given slopes in Opticon™ 2 Monitor software. Real-time RT-PCR efficiency rates were high . Experiments were performed three times in triplicate, the inter-test variation was ≤ 2%, and the intra-test variation ≤ 1%.To optimize the real-time reverse transcription (RT)-PCR conditions for quantification, the optimal MgClDuring human embryonic development from gw 8 to gw 12, basement membrane zones of the developing skin stained positive for COMP whereas the mesenchyme remained unstained Figure . In limbin situ hybridization, the mRNA for COMP was detected in the cytoplasm of chondrocytes of the superficial and middle zones of healthy cartilage tissue (data not shown) and also in chondrocytes mainly found in clusters in the area adjacent to the main defect in OA cartilage . In contrast, in both cell types found in the areas adjacent to the main defect of OA tissue, approximately 320 gold particles (SEM = 13.4) were detected , approximately 83% difference in staining intensity for the cells taken from the two areas.The number of gold particles detected in the samples with a macroscopically normal appearance from OA tissue revealed staining intensities of approximately 42 (SEM = 3.4) in type 1 cells and 66 (SEM = 4.1) in type 2 cells. This represents a significant difference  [COMP expression should be downregulated by FBI-1. As shown here, in late stages of human OA in vivo, chondrocytes upregulate their COMP expression and, as shown earlier, downregulate their collagen type II expression [COMP and collagen type II [COMP and collagen type II in human OA, which in turn enhances the matrix breakdown and thereby increasing the mechanical load of the diseased tissue, this mechanical load could counteract the LRF/FBI-1 effect and upregulate only the COMP expression in late stages of the disease, as shown here for the areas bearing the highest load in human OA tissue in vivo.One of the known factors of in vitro . The hum (FBI-1) . If FBI- (FBI-1)  acts as pression . This di type II . If LRF/In summary, our results demonstrate that COMP is present in the earliest stages of human bone and joint development. COMP is also a component of the adult healthy articular cartilage matrix and is produced by the chondrocytes. Furthermore, we were able to show that during late stages of OA, increased amounts of COMP are produced by type 1 and type 2 cells in the area adjacent to the main defect and that due to its wide binding repertoire, COMP might therefore be involved in the regeneration efforts of OA cartilage tissue as a factor secreted by chondrocytes to ameliorate the matrix breakdown.AER = apical ectodermal ridge; COMP = cartilage oligomeric protein; DIG = digoxigenin; FBI-1 = factor binding inducer of short transcripts protein-1; gw = gestational week; IgG = immunoglobulin G; LRF = leukemia/lymphoma-related factor; OA = osteoarthritis; PBS = phosphate-buffered saline; RT-PCR = reverse transcription-polymerase chain reaction.The authors declare that they have no competing interests.in situ hybridization of the normal and OA cartilage. MK is responsible for the Western blots. SK and NM are responsible for the real-time PCR and the overall editing of the manuscript. All authors read and approved the final manuscript.TSC performed the immunohistochemistry and


Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits.Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro independent of VEGF-A.Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.Our  The development and continuous remodeling of the skeleton demand a tightly regulated balance between the bone-forming and bone-resorbing processes. One of the key components of bone remodeling is constant development of vasculature. Vasculature is required for transport of nutrients and precursor cells, such as precursors of chondroclasts and osteoclasts, to the renewing bone tissue. Angiogenesis has also been shown to be crucial for the replacement of cartilage by bone during skeletal growth and regeneration. Vascular endothelial growth factor-A (VEGF-A), produced by hypertrophic chondrocytes during endochondral bone formation, stimulates controlled invasion of chondroclasts into the cartilage ,2. VEGF-Invasion of osteoclasts into hypertrophic cartilage requires presence of VEGF-A  which biEndostatin, a known antagonist for VEGF-A, is 20 kDa C-terminal fragment of collagen XVIII found in basal membranes. Endostatin, that binds to αv- and α5-integrins , inhibitin vitro. In order to investigate the direct effects of these angiogenic and antiangiogenic substances on osteoclast mediated bone resorption, we used classical resorption pit assay and osteoclast differentiation assay of bone marrow hematopoietic stem cells. We were, for the first time, able to demonstrate that endostatin has a significant regulatory role on both osteoclastic bone resorption and osteoclastogenesis in vitro.Aim of our study was to further define possible role of endostatin and VEGF-A on osteoclasts et al. and by Chambers et al. [2 or 0.5 cm2). After 30 minutes, the nonattached cells were rinsed away, and the remaining cells on the bone slices were cultured in Dulbecco's modified Eagle's medium (α-MEM) buffered with 20 mM HEPES and containing 0.84 g of sodium bicarbonate/liter, 2 mM L-glutamine, 100 IU of penicillin/ml, 100 μg of streptomycin/ml and 7–10% heat-inactivated fetal calf serum (FCS), at +37°C (5% CO2 and 95% air). The cells were divided into ten groups: the control group had α-MEM-FCS as their culture medium with no added substances (later referred as control), the VEGF-A-treated cells had 100 ng/ml, 50 ng/ml or 10 ng/ml VEGF-A in α-MEM-FCS (later referred as VEGF). The endostatin groups had 0.02, 0.2 or 2 μg/ml endostatin in α-MEM-FCS (later referred as ENDO) and the last groups had both VEGF-A and endostatin added into the complete culture media (later referred as VEGF+ENDO). The last groups had 100 ng/ml VEGF-A and 2 μg/ml endostatin, 50 ng/ml VEGF-A and 0,2 μg/ml endostatin or 10 ng/ml VEGF-A and 0,02 μg/ml endostatin. The cells were allowed to grow for 48 h, after which the bone slices were fixed with freshly made refrigerated 3% paraformaldehyde (PFA) and 2% sucrose in phosphate-buffered saline (PBS) for 10 minutes at room temperature. The data shown in this manuscript is gathered from three independent experiments, each of which gave identical results.The procedure for the isolation and culture of osteoclasts described earlier by Boyde s et al. ,21 was ms et al. . Briefly2) at a concentration of 1 × 106 cells/well. Cells on the bone slices were cultured in four groups: control, VEGF (100 ng/ml) and ENDO (2 μg/ml) and VEGF + ENDO respectively. The control group was cultured in α-MEM medium  with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamin, 100 IU/ml penicillin, 10 μg/ml streptomycin and 20 mM Hepes which medium will be later referred to as α-MEM-FCS. Cells were cultured in 500 μl α-MEM containing 30 ng/ml RANKL  and 10 ng/ml M-CSF (R&D Systems). Half of the medium was replaced every 3rd day. Cells were cultured at +37°C  for 7 days, after which the cultures were stopped by fixing the cells with 3% paraformaldehyde (PFA)/2% sucrose in PBS.The procedure for osteoclast differentiation described earlier by Takahashi et al.  was sligBone cells were cultured for 48 hours in resorption pit assay and 7 days in the differentiation assay. After the culture period, bone slices were fixed as described above. The cells were stained for tartrate-resistant acid phosphatase (TRACP), a commonly accepted marker of osteoclasts  using a 2O2. Morphometric analysis of the resorption pits was performed with an MCID image analyzer, utilizing M2 software .Prior to the staining of the pits, total detachment of the cells from the bone slices was ensured by wiping the cellular surface of the slices with a soft brush. The pits were stained with peroxidase-conjugated wheat germ agglutinin-lectin  for 20 minutes, washed with PBS, and incubated for 5 minutes in diaminobenzidine  +0,03% HData presented are the results of at least two independent experiments. Values are the mean ± SEM. Significance was calculated using one-way ANOVA followed by Student's t-test with statistical package Origin 6.0. The p-values less than 0.05 were considered as significant.The number of osteoclasts after 48 hours in each sample group was counted by analyzing multinuclear TRACP-positive cells in the resorption assay. No significant differences in the number osteoclasts were found between the different groups Fig  indicatiThe total resorbed area was significantly increased in response to VEGF-A treatment and the effect was dose dependent Fig. . EndostaIn the presence of RANKL and M-CSF the bone marrow hematopoietic cells differentiate into osteoclasts which can be recognized by positive TRACP-stain and multinuclear appearance. Since the basal resorption was not affected by endostatin in the pit assay, we conducted an assay to investigate if endostatin could inhibit the differentiation of osteoclasts. We added VEGF-A to the medium containing M-CSF and RANKL in order to achieve maximal differentiation stimulus Fig. . EndostaBone remodeling is initiated by recruitment of osteoclast precursor cells from bone marrow or circulating monocytes. Homing of these precursors through the vascular wall is required for successful bone turnover. Intimate contact of the bone remodeling site to adjacent vascular sinusoids indicates interplay between the vascular endothelium and the bone cells. Co-culture of osteoblasts and endothelial cells stimulate the osteoblasts to secrete VEGF-A and to express RANKL, which is crucial for osteoclastogenesis . VEGF-A ex vivo metatarsal model in which fusion and migration of osteoclast precursors is required for the osteoclastic bone resorption [in vivo [in vitro, mature osteoclasts already exist. Still, we see a clear reduction of VEGF-A-stimulated resorption in the endostatin treated pit assays. Thus, it is possible that endostatin may not only limit the migration of osteoclast precursors, but it may also inhibit the VEGF-A-induced osteoclast activation. Pufe et al. have detected similar influence of endostatin on hyaline articular cartilage. Endostatin alone had no effect on basal levels on the phosphorylation of ERK1/2, whereas co-incubation with endostatin blocked VEGF-induced ERK1/2 phosphorylation in immortalized human chondrocytes (C28/I2) [Significance of endostatin in bone has been demonstrated elegantly in the sorption . Osteocl[in vivo . In our (C28/I2) ,35.Osteoclast precursor differentiation into mature osteoclasts is disturbed in the absence of VEGF-A . VEGF-A in vitro. RANKL and VEGF-A stimulate the osteoclast recruitment through the same signaling pathway [Combination of RANKL, M-CSF, and VEGF-A activated intense differentiation of osteoclasts in our study  pathway  and thesin vitro experiments suggesting that regulatory role of endostatin on osteoclasts may not be critical. In pathological conditions, however, endostatin may have a significant position. Endostatin is normally found in serum [in vitro data support the possibility that endostatin could be used as a physiological agent to inhibit the osteoclast activity in inflammatory arthritis. Further in vivo studies in animal models may be considered to explore that possibility.Endostatin did not inhibit basal bone resorption in our in serum ,37 and ain serum -40. Endoin serum . Our in in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.Our The author(s) declare that they have no competing interests.AS, KN and JI have performed the cell cultures and their examination including the statistical analysis and together with TH and JT participated in development of the experiments. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The HIV/AIDS epidemic continues to spread despite more than 20 years of significant research and major advances in its treatment. The introduction of highly active antiretroviral therapy in recent years has significantly improved disease treatment with a dramatic impact in HIV/AIDS associated morbidity and mortality in countries which have access to this therapy. Despite these advances, such therapies are imperfect and other therapeutic modalities, including immune-based therapies, are being actively sought. Potential benefits of immune-based therapies include: 1) the improvement of HIV-specific immunity to enhance control of viral replication, 2) the improvement of other aspects of host immunity in order to prevent or delay the development of opportunistic infections and 3) the potential to purge virus from cellular reservoirs which are sustained despite the effects of potent antiretroviral therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied as one of these immune-based therapies. Several randomized, controlled trials have demonstrated benefits of using GM-CSF as an adjunct to conventional anti-retroviral therapy, although such benefits have not been universally observed. Individual studies have shown that GM-CSF increases CD4+ T cells counts and may be associated with decreased plasma HIV RNA levels. There is limited evidence that GM-CSF may help prevent the emergence of antiretroviral drug resistant viruses and that it may decrease the risk of infection in advanced HIV disease. Despite its high costs and the need to be administered subcutaneously, encouraging results continue to emerge from further studies, suggesting that GM-CSF has the potential to become an effective agent in the treatment of HIV infection. More than 20 years after its discovery, and despite extensive research in the field, HIV-1 infection remains one of the most important public health problems in the world. The HIV/AIDS epidemic continues to spread and an increasing number of people continue to live with HIV/AIDS and die from it. The advent of highly active antiretroviral therapy (HAART) marked a cornerstone in HIV/AIDS treatment that drastically changed the prognosis of HIV infection, by its ability to induce sustained suppression of viral replication . Yet HIVin vitro to enhance the activity of the antiretroviral agent zidovudine (AZT) in macrophages [In vitro, the presence of GM-CSF suppresses the expression of CXCR4 mRNA and CCR5 mRNA by monocytes differentiating in macrophages, resulting in macrophages that are relatively resistant to M-tropic HIV infection [GM-CSF is a pleiotropic growth factor that enhances the number and function of various cells from both the myeloid and lymphoid lineages, including neutrophils, monocytes and lymphocytes . It is orophages ,23 and trophages . In vitrnfection .in vitro studies that have suggested that GM-CSF enhances the action of anti-retroviral drugs (ARVD) in macrophages [in vivo. There appears to be lower frequency of resistant-mutations among subjects on zidovudine and GM-CSF, as part of their anti-retroviral regimens, versus those on AZT alone [in vitro findings and early in vivo studies, several randomized controlled trials were developed that studied the effect of GM-CSF as a treatment for HIV-1 infected individuals.In addition to rophages ,23, dataZT alone . This fiZT alone -28, the ZT alone -31. Foll10 copies/ml in the placebo group compared with 4.21 log10 in the treatment group (p = 0.29) and the mean CD4+ T cells count in the placebo group was 243 cells/mm3 compared with 178 cells/mm3 in the GM-CSF group. All subjects were on stable antiretroviral therapy, including either indinavir or ritonavir, for a mean period of 5.0 months in the placebo group and 4.8 months in the treatment group. They received either 250 μg of GM-CSF or placebo subcutaneously 3 times per week for a total of eight weeks. All subjects were followed closely every two weeks during the study and twice at week 3 and week 5 after the study ended. During the study and at both follow up time points, the viral load remained within 0.5 log10 copies/ml of the baseline values for both groups.Few randomized controlled trials of GM-CSF have shown a clear, significant reduction in HIV replication. The first randomized controlled trial on the use of GM-CSF in non-neutropenic HIV-1 infected subjects, published in 1999, did not show any significant effect of GM-CSF on plasma HIV RNA levels . This tr10 from baseline (50% vs. 10%). Since this size of a viral load decrease has been associated with clinical benefits, this study suggests that GM-CSF may have a beneficial effect in a subset of individuals.Despite no overall changes in the mean HIV RNA load between groups, more subjects in the GM-CSF group than in the control group had HIV RNA values decreased by >0.5 log3 in the control group and 50.8 cells/mm3 in the GM-CSF group. The majority of subjects entered the study with a viral load over 30000 copies/ml (62% for the placebo arm and 63% for the GM-CSF arm). There was no significant decrease in HIV-RNA in the combined strata in either the placebo or treatment group. However, GM-CSF had a positive influence on other viral parameters. GM-CSF use delayed virologic failure in those patients with plasma HIV RNA levels less than 400 copies/ml before initiation of GM-CSF therapy. At 6 months, 24 out of 29 (83%) of subjects on GM-CSF maintained viral loads below the limit of detection compared to 15 out of 28 (54%) of those on placebo (p = 0.02). This, in turn, reduced the need for antiretroviral regimen change. In this trial, ARVD regimen changes were allowed, which could have obscured a preferential decrease in viral load by GM-CSF. As such, there were fewer changes in ARVD regimens in the GM-CSF group (19%) than in the placebo group (38%) for the lower viral load stratum (p = 0.03). In the higher stratum, no significant difference in treatment change was observed . Again, this supports the idea that low-dose GM-CSF may have the potential to limit HIV replication and prevent or delay the development of drug resistant viruses, as described earlier in in vitro and in vivo studies.In the largest double blind, randomized controlled trial on GM-CSF use in HIV infected individuals published to date, 309 subjects stratified according to viral load (≤ 30000 copies/ml vs. > 30000 copies/ml) received either 250 μg of GM-CSF or placebo three times per week for 24 weeks . In tota3. Patients were excluded if they had an active AIDS defining diagnosis at the time of randomization or if they had been exposed to zidovudine for >6 months prior to study entry. The mean HIV RNA plasma levels at baseline were 93000 copies/ml in the placebo group and 155000 copies/ml in the GM-CSF group (p = 0.21). All the subjects received AZT, and 65% and 68% of subjects in the placebo and GM-CSF group respectively were also on a second agent, either ddI, ddC, 3TC or Saquinavir. Prior opportunistic infection rates were 58% in the placebo group and 70% in the treatment group (p = 0.14). This study did show a statistically significant effect of GM-CSF on viral loads. Mean HIV RNA levels declined in the GM-CSF group throughout the 6 months of the study. Over this period, the change was -0.07 log10 copies/ml in the control group as opposed to -0.60 log10copies/ml in the treatment group . As well, there was a greater number of subjects in the GM-CSF group with a decrease of 1 log10 or greater in viral load  compared with the placebo group  (p = 0.02). The reason why a decrease in viral load was observed in this study and not in other trials is unclear. It was the only trial with a smaller dose of GM-CSF  and all patients were receiving AZT, both of which might have played a role in this difference.In a Brazilian study, 105 individuals with AIDS were enrolled in a placebo-controlled, double-blind randomized control trial to receive AZT along with GM-CSF (125 μg) or placebo twice weekly for 6 months . Subject10 copies/ml) for at least 7 days prior to entry and where no HAART regimen change was allowed during the 16 weeks period of the trial. Subjects were divided in 2 groups, depending if their CD4+ T count was below or above 200 cells/mm3 at baseline and then randomized to either 250 ug of GM-CSF or placebo three times per week for 16 weeks. All patients subsequently received a 32-week course of open label GM-CSF. Baseline characteristics were similar in both groups. At baseline, in the ≥ 200 and <200 CD4+ cells/mm3 strata, median plasma RNA levels were 3.81 log10 and 4.46 log10 copies/ml, with no difference between control and treatment groups. After the 16 weeks of double-blinded treatment, the change in HIV RNA levels was +0.048 log10 copies/ml in the GM-CSF group compared with -0.103 log10 copies/ml (p = 0.036) in the placebo group, both strata combined. However, when the two strata (≥ 200 and <200 CD4+ cells/mm3) were studied individually, the changes in mean viral loads were not significant. Thus, in this trial, subjects in the GM-CSF group, irrespective of their initial CD4+ count, tended to have a modest increase in HIV RNA levels at the end of the 16-week randomized period. Although the modest increase in viral load was significant, it was not associated with a decrease in CD4 counts or an increase in clinical events, as is discussed later.More recent clinical data on the use of GM-CSF in combination with HAART continues to show some effect of GM-CSF on viral load . These d3. In both groups the viral load peaked at 6 weeks and trended down afterwards. In the GM-CSF group, the maximum viral load reached a mean of 4.97 log10 compared with 5.54 log10 in the scheduled treatment interruption-only (STI-only) group (p = 0.03). Over a period of twelve weeks, the mean area under the curve for viral loads were 47.77 log10 in the GM-CSF group and 51.88 log10 in the STI-only group (p = 0.07) This suggests not only that there is no deleterious effect of GM-CSF on plasma HIV RNA levels but that GM-CSF may help control the viral load in patients who need to stop HAART for a short period.Finally, a Swiss study evaluated the use of GM-CSF 300 μg three times a week for the first four weeks of a 12-week HAART interruption period . This smet al. is not clear. This study was somewhat unique in that it included only patients with uncontrolled viral replication. It appears likely that the impact of GM-CSF on viral load is dependant upon the setting in which GM-CSF is used. Furthermore, GM-CSF may selectively enhance the antiviral activity of specific antiretroviral agents (e.g. AZT). Also, as may be becoming apparent with other immune-based therapies, the greatest effect of GM-CSF may be observed in situations where there is the greatest degree of virologic suppression and associated immunologic recovery.Overall, the evidence regarding the effect of GM-CSF on plasma HIV-1 RNA levels is somewhat conflicting. Four of the five trials reviewed show either a significant decline or no statistical changes in the viral load. The explanation for the observed increase in viral load in the GM-CSF group in the trial by Jacobson 3 and 57 ± 58.9 cells/mm3 in the control group (p = 0.02). A significant majority (70%) of subjects treated with GM-CSF demonstrated an increase of >30% of their CD4+ T cell counts over baseline at any given time versus a minority (30%) in the placebo group (p = 0.07). When those patients with baseline CD4+ T cell counts of <50 cells/mm3 were excluded from the analysis, in order to ensure an increase of >30% was not due to daily variability, 6 of 7 patients in the GM-CSF group and 1 of 8 patients in the placebo group had a CD4+ T cell increase of >30% (p = 0.01). This may have a clinical impact as a >30% increase of the CD4+ T cell count in light of a stable viral load has been associated with a relative risk reduction of disease progression in a previous study [The initial randomized control trial of GM-CSF use in non-leukopenic HIV infected individuals, referred to previously, reported other important findings. CD4+ T cell counts reached higher levels in the treatment group, but these results did not reach statistical significance . The meaus study .An earlier randomized controlled study looking into the effect of GM-CSF on leukopenia in HIV-infected individuals also demonstrated an increase in CD4+ T cell counts. After 12 weeks of therapy (300 μg GM-CSF daily for 1 week then 150 μg twice-a-week for 11 weeks), absolute CD4+ T cell counts rose by 53% compared to baseline (p < 0.001) and was statistically different than that observed in the control group (p < 0.001) .et al, the authors reported a modest increase in the CD4+ T cell count in both groups at six months [3 compared with 12 cells/mm3 in placebo group (p = 0.42). As with the study by Skowron et al, they also observed a significant difference in the number of subjects who had a ≥ 30% increase of the CD4+ T cell count. Only 59% of subjects in the placebo group achieved this increase as opposed to 80% in the GM-CSF group (p = 0.03).Other trials observed a trend towards modest, non-significant, increases in the absolute CD4+ counts. In the study by Brites x months . In the et al., the authors reported a change in the CD4+ T cell count of +29 cells/mm3 in the GM-CSF vs. -8 cells/mm3 in the placebo group for the stratum of subjects with >200 CD4+ T cells/mm3 at baseline (p = .20) [3 stratum, with +5 cells/mm3 in the GM-CSF group at 16 weeks vs. -5 cells/mm3 in the placebo group but this did not reach statistical significance (p = 0.22).In the other, more recent trial, from Jacobson p = .20) . They ob6 cells/L for the placebo group and 50.8 × 106 cells/L for the treatment group. At 12 months the mean CD4+ T cell count was 102 ± 15 × 106 cells/L in the placebo group vs. 152 ± 18 × 106 cells/L in the treatment group. This was also reflected by statistically significantly greater increases in the CD4+ T cell count at 1, 3 and 6 months in the GM-CSF group.Fairly convincing evidence of a significant increase in absolute CD4+ leukocyte count following treatment with GM-CSF comes from the phase III randomized control trial by Angel and colleagues, described earlier . The basIt might be speculated that, since this was the largest (n = 307) and one of the longest (24 weeks) randomized control trial of GM-CSF use in HIV infected individuals, it maybe the only study with enough power to demonstrate statistical significance. As other randomized control trials show trends towards higher CD4+ T cell counts in the GM-CSF group, and statistically significant difference in various sub-analysis, it is possible that an appropriately conducted meta-analysis would clarify the impact of GM-CSF on CD4+ T cell counts.6 cells/L at baseline to 537 × 106 cells/L at four weeks after stopping HAART in the STI-only group (p < 0.001). In the GM-CSF treated group, there was no significant change in the CD4+ T cell counts four weeks after stopping HAART. CD4+ T cell counts were 890 × 106 cells/L at baseline and 792 × 106 cells/L at week four (p = 0.6). This adds evidence that GM-CSF could have a beneficial effect on CD4+ T cell counts.The recent study by the Swiss group also supports a positive effect of GM-CSF on CD4+ lymphocytes count . In thatet al, a total of 4 patients had to discontinue GM-CSF use because of toxicity or acute allergic reactions [GM-CSF has an excellent safety and tolerability profile when used in HIV-1 infected individuals ,15-17. Ieactions ,15,16. Tet al. has been the only study to use clinical events as endpoints, using the Centers for Diseases Control and Prevention definition of opportunistic infections (OI), bacterial pneumonia or death as their primary endpoint [The large phase III trial by Angel endpoint . An effeet al. did noticed that all 17 subjects in the GM-CSF arm who developed an OI had a prior history of one or more of these infections. In the placebo group, only 50% of the 14 subjects who developed an OI during the study had a prior history of OI (p < 0.01). This might prove to be an important role for adjunct treatment with GM-CSF as OI are still an important cause of morbidity and mortality in HIV infected individuals.For individuals who do not have a history of OI, GM-CSF may decrease the risk of a first opportunistic infection. Despiteet al. did show a non-significant reduction in clinical events in the GM-CSF group [The most recent randomized control trail by Jacobson SF group . No HIV et al. failed to show any impact of GM-CSF on clinical events during HAART interruption. However, they studied only 33 patients with high CD4 counts and off HAART for a limited period of time [Finally, the study by Fagard  of time .Despite all these results, questions still remain as to whether use of GM-CSF is associated with a reduction in the incidence of AIDS related morbidity and mortality, as even the authors of the largest phase III study published to date admit to a lack of power in their trial . The intIn various studies GM-CSF has a positive effect on important parameters of HIV infection, namely plasma HIV RNA levels and CD4+ lymphocytes counts. Although the positive effects are modest and not universally observed, they are significant in many trials. Moreover, the positive effect on these measures may translate into significant clinical benefits. Clinical outcome results of current randomized controlled trials are, thus far, somewhat encouraging. Despite the frequent lack of statistical significance, there are positive trends towards clinical benefit of GM-CSF use in these studies. Moreover, the largest randomized control trial did show that GM-CSF produces a significant reduction in the time to first infection or death. The possibility of allowing longer disease free periods is a desirable outcome for HIV infected individuals, contributing to improved quality of life. However, the high cost of GM-CSF and its mode of administration may be difficult hurdles for patients to overcome.Future trials designed to look at specific clinical outcomes, for example diseases free period, progression of HIV infection and quality of life, might bring to light additional beneficial effects of GM-CSF. This would require focusing on patients with advanced HIV disease and lower CD4 counts. Alternatively, future trials could focus on the use of GM-CSF as an adjuvant therapy, either to HAART or as an adjuvant with HIV or other vaccines. There is growing evidence that GM-CSF enhances the immune response to vaccines directed against both infectious agents and various cancers . ClinicaIt remains to be seen if GM-CSF will ever loose its experimental status and become an accepted therapy for selected individuals HIV infection. The evidence for the role of immunotherapy in HIV/AIDS is ever increasing and GM-CSF might very well become a widely accepted treatment in the years to come.The author(s) declare that they have no competing interests.Table 1 is a summary of clinical trials of GM-CSF in the treatment of HIV infection.Click here for file